Experimental Gerontology 39 (2004) 1267–1276
www.elsevier.com/locate/expgero
Aging in a very short-lived nematode
Michael P. Gardnera,*, David Gemsb, Mark E. Vineya
a
School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK
Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK
b
Received 4 May 2004; received in revised form 17 June 2004; accepted 18 June 2004
Available online 8 August 2004
Abstract
Aging has been characterised in detail in relatively few animal species. Here we describe the aging process in free-living adults of the
parasitic nematode Strongyloides ratti. We find that the phenomenology of aging in S. ratti free-living females, resembles that of the shortlived free-living nematode Caenorhabditis elegans, except that it unfolds far more rapidly. The mean (3.0G0.1 days) and maximum (4.5G
0.8 days) lifespans of free-living S. ratti females are approximately one quarter of equivalent values for C. elegans. Demographic senescence
(a hallmark of aging) was observed in free-living S. ratti, with a mortality rate doubling time of 0.8G0.1 days (females), compared with
2.0G0.3 in C. elegans. S. ratti lifetime fertility and lifespan were affected by temperature, and there is an age-related decline in feeding rate
and movement, similar to C. elegans, but occurring more quickly. Gut autofluorescence (lipofuscin) also increased with age in S. ratti freeliving females, as in aging C. elegans. These findings show that the extreme brevity of life in free-living S. ratti adults, the shortest-lived
nematode described to date, is the consequence of rapid aging, rather than some other, more rapid and catastrophic life-shortening pathology.
q 2004 Elsevier Inc. All rights reserved.
Keywords: Strongyloides ratti; Aging; Phenotypic plasticity; Lifespan; Fecundity
1. Introduction
In this study, we describe the first stage of a full
characterisation of aging in the nematode Strongyloides
ratti. By studying this organism, insight may be gained into
the extent of variation in the lifespan of animal species and of
phenotypic plasticity in aging and into the evolution of
longevity and aging.
The life-cycle of S. ratti contains both free-living and
parasitic stages (Fig. 1). The parasitic stage is female only
and lives in the gut of its rat host and reproduces by
mitotic parthenogenesis (Viney, 1994), passing eggs via
the faeces into the external environment. The eggs can
develop either directly into infective third stage larvae
(iL3s) or into free-living adult females and males (Fig. 1).
These adults mate by conventional sexual reproduction
and produce progeny which develop into iL3s and infect
* Corresponding author. Tel.: C44-117-928-7470; fax: C44-117-9257374.
E-mail address: mike.gardner@bristol.ac.uk (M.P. Gardner).
0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.exger.2004.06.011
new hosts (Viney et al., 1993). Anecdotal observations
have noted a large difference in the lifespan potential of
the adult free-living and parasitic female morphs
(approximately 1 week and 1 year, respectively) (Viney,
1994; Gemmill et al., 1997). Given that these two adult
morphs are genetically identical (Viney, 1994), such a
difference in survival is potentially a consequence of
differences in gene expression alone.
Why study aging in S. ratti? Comparative studies of
aging in different animal species can provide basic
information about the forms of aging that are biologically
possible. For example, studies of the Cnidarians
Hydra vulgaris (Martı́nez, 1998) and Cereus pedunculatus
(Comfort, 1979) suggest that neither of these species
senesce. Among animals that age, there is great variation
in longevity (Finch, 1990). For example, among mammals,
maximum lifespans range from around 3 years in the mouse,
Mus musculus, to an estimated 211 years in the bowhead
whale, Balaena mysticetus (George et al., 1999). More
striking variation in aging exists in invertebrate taxa. For
example, estimates of maximum lifespans in nematode
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M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
Fig. 1. Life-cycle of Strongyloides ratti with two discrete developmental switches, shown as grey boxes: (1) a sex determination event; (2) a female only
developmental switch. Taken from Harvey et al., 2000. Also included are larval stages L1–L4. The life-cycle contains both parasitic and free-living
generations; an adult parasitic female (top) and free-living adult female (bottom). BarsZ200 mM.
species range from 7.9 days in the terrestrial nematode
Mesodiplogaster biformis (Sohlenius, 1969) to 20 years in
the parasitic nematode Loa loa (Gems, 2001).
Studies that relate species’ ecology to aging have also
been used to interrogate the evolutionary theory of aging.
The kernel of this theory is that due to the forces of extrinsic
mortality (e.g. starvation, predation, disease), the distribution of the probability of reproducing will be skewed to
earlier ages, even in a theoretically non-aging organism.
A predicted consequence of this is that the force of natural
selection against alleles with late-acting deleterious effects
will be less than those with earlier-acting effects (Williams,
1957). The resulting accumulation of alleles with
late-acting, deleterious effects (and perhaps early acting,
fitness-enhancing effects) is the cause of aging. From this,
the maximum lifespan which any species has evolved is
predicted to have some correspondence with the level of
extrinsic mortality typically experienced by that species in
its environment (Edney and Gill, 1968).
This prediction has been supported by a growing number
of comparative studies of aging. For example, comparisons
between rodents and similar sized bats show increased
lifespan in the latter (Austad and Fischer, 1991); the
increased longevity of bats is believed to be the consequence of the reduction in extrinsic mortality resulting from
the capacity for flight, which reduces predation (Wilkinson
and South, 2002). Similarly, Virginia opossums (Didelphis
virginiana) from an island population with reduced
exposure to predators, exhibited delayed aging compared
with a control, mainland population (Austad, 1993). In
eusocial organisms, lower mortality rates experienced by
certain morphs results in evolution of greater longevity in
those morphs, e.g. in honeybees (Page and Peng, 2001), ants
(Chapuisat and Keller, 2002) and naked mole rats (Sherman
and Jarvis, 2002).
Many animal species also show phenotypic plasticity in
aging, of which two types may be distinguished. The first is
the relatively limited response to a changing environment
by a given stage in the life-cycle. An example of this is
experimental caloric restriction, which extends lifespan in
numerous species, including the nematode C. elegans
(Klass, 1977; Lakowski and Hekimi, 1998), the fruitfly
M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
Drosophila melanogaster (Nusbaum and Rose, 1999),
rodents (McCay et al., 1935) and, probably, primates
(Roth et al., 2002). Another example is the effect of
diapause on aging; for example, in the Turkish hamster,
Mesocricetus brandti, hibernating animals lived significantly longer than those not hibernating (Lyman et al.,
1981).
A more dramatic form of phenotypic plasticity in aging
is associated with the development of different morphs
within a given life-cycle. For example, in C. elegans,
there is a facultative diapausal form of the third stage
larva, the dauer larva, which can survive for up to
3 months (Klass and Hirsh, 1976). This compares to a
two-to-three week lifespan in C. elegans adults (Klass,
1977). Striking variation between lifespan in adult morphs
also occurs in social insects (Chapuisat and Keller, 2002).
Understanding the evolutionary and physiological determinants of such plasticity can provide insight into the
biology of aging.
The aims of the present study are to characterise
aging in free-living adult females and males of S. ratti
and to determine the factors that affect their lifespan. In
particular, a major question is whether the reported short
lifespan of Strongyloides spp. and of related species such
as Rhabdias bufonis (Yamada et al., 1991; Spieler and
Schierenberg, 1995) is actually a consequence of rapid
aging, rather than extrinsic, environmental causes, or
some form of major defect leading to death. In
C. elegans, lifespan is reduced by the Escherichia coli
bacteria on which they are usually cultured (Gems and
Riddle, 2000; Garigan et al., 2002; Garsin et al., 2003),
by higher temperatures (Klass, 1977), mating between the
sexes (Gems and Riddle, 1996) and attempted mating
between males (Gems and Riddle, 2000). We have tested
the effect of these factors on lifespan in S. ratti, and in
so doing optimised culture conditions for survival, in
order to maximise the contribution of intrinsic senescence to lifespan and to minimise that of deleterious
environmental factors. We then asked whether free-living
S. ratti adults cultured under optimised conditions exhibit
demographic aging, i.e. an age-dependent mortality rate
acceleration, a hallmark of aging (Wachter and Finch,
1997). In C. elegans, there is an increase in autofluorescence with age (Davis et al., 1982; Garigan et al.,
2002), similar to accumulation of age-pigment (lipofuscin) seen during mammalian aging. We have tested
whether this occurs in free-living S. ratti adults.
We find that with culture conditions optimised for
survival, S. ratti free-living adults exhibit a Gompertzian
acceleration of age-specific mortality. By comparison with
C. elegans, this acceleration was more rapid (indicating
faster aging) and the initial mortality rate (IMR) was greater
(implying greater frailty). Thus, the S. ratti free-living
morph is the shortest lived and most rapid-aging nematode
described to date.
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2. Materials and methods
2.1. Nematodes and bacteria
The S. ratti isofemale line, ED321 Heterogonic (Viney,
1996), was maintained by serial passage in female Wistar
rats (B and K Universal) or female nude rats (RNU-rnu)
(Harlan, UK). Food and water was provided ad libitum and
nude rats were maintained on water containing 0.01% (v/v)
Baytril (Bayer, UK), a broad spectrum antimicrobial agent.
Infections were initiated by sub-cutaneous injection of
1000 infective third stage larvae (iL3s) and faecal material
was collected from rats overnight in grid-bottomed cages
and was cultured and maintained at 19 8C using standard
methods (Viney, 1994). C. elegans wild-type laboratory
strain, Bristol (N2), E. coli OP50 and streptomycin
resistant E. coli OP50 (E. coli SR) bacterial food sources
were obtained from the Caenorhabditis Genetics Center
(CGC). C. elegans was maintained on NGM agar at 19 8C
and seeded with E. coli OP50 as a food source (Sulston and
Hodgkin, 1988), unless otherwise stated.
2.2. Culture conditions for free-living S. ratti
L4 female and male S. ratti worms were taken from
2-day-old faecal cultures of infected rat faeces, cleaned to
remove associated bacteria by repeated washing in sterile
distilled water and cultured on NGM plates under optimised
culture conditions (400 mg/ml streptomycin, E. coli SR),
unless otherwise stated. Lifespan was measured as
described for C. elegans (Klass, 1977). Briefly, worms
were transferred daily to fresh plates and the number of
survivors scored. Cultures were maintained at 19 8C and 10
L4 S. ratti females were placed on each plate (100 worms
per treatment), unless otherwise stated. Kaplan–Meier
analysis was used to compare mean lifespan between
treatments using a log-rank c2 test.
The lifespan of virgin female S. ratti under optimised
culture conditions (400 mg/ml streptomycin, E. coli SR) was
compared with lifespans on non-proliferating bacteria. Nonproliferating bacteria were produced, as previously
described (Garigan et al., 2002). Briefly, E. coli OP50 was
seeded onto NGM plates and allowed to grow for 2 days
at room temperature before the antibiotics were added;
20 mg/ml kanamycin or 200 mg/ml streptomycin as bactericidal antibiotics and 12.5 mg/ml tetracycline as a bacteriostatic antibiotic. Cultures of Bacillus spp. or Streptococcus
faecalis isolated from rat faeces were identified using
Macconkey and blood agar indicator plates, and Gram
staining (Cowan et al., 1993). Nematodes were maintained
in 60 mm Petri dishes, except for single worms in
population density measurements, which were maintained
in 35 mm dishes. All statistical analyses were performed
using the JMP statistical analysis package (version 5.0, SAS
Institute, Inc, Cary, NC, USA), with level of significance
tested at a level P!0.05.
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2.3. Analysis of age-specific mortality
Survival of virgin free-living S. ratti females was
measured under optimised culture conditions (400 mg/ml
streptomycin, E. coli SR) and a temperature of 25 8C
(optimal for fertility). C. elegans survival was measured
under two conditions: (i) S. ratti optimised conditions to
which was added 60 ml of the supernatant in which the
S. ratti worms were washed, (the latter introduced low
levels of contaminating microbes, assumed to be present
under optimised S. ratti culture conditions, allowing
comparison of survival of S. ratti and C. elegans under
near identical culture conditions); (ii) monoxenic conditions consisting of NGM plates with E. coli SR but with
no added supernatant. 25 worms per plate and 500 worms
per treatment were used, and mortality scored approximately every 8 h for S. ratti and every 12 h for C. elegans,
until all animals were dead. Mortality data were fitted to
the Gompertz equation, m(t)ZAeat, where m(t) is the
mortality rate at time t, A is the IMR and a is the Gompertz
exponential function (Finch, 1990). The occurrence of an
age-specific mortality rate acceleration is a definitive
feature of aging (Finch, 1990). From plots of ln (mortality
rate) against age, we calculated A, the IMR, the mortality
rate doubling time (MRDT) and estimated median (time
for 50% survival) and maximum lifespans and their
standard errors. Mortality parameters for S. ratti and
C. elegans were compared using Student’s t-tests.
2.4. Fecundity analysis
Free-living S. ratti females and males were maintained
under optimised culture conditions (400 mg/ml streptomycin, E. coli SR). Gravid free-living females were allowed to
lay eggs, and transferred daily to fresh plates and the
number of eggs were recorded. These were allowed to hatch
over a 2-day-period and the number of resulting larvae (i.e.
viable egg production) counted, and daily and total
fecundities calculated over a range of temperatures (4, 10,
15, 19, 25, 30 and 37 8C). We tested whether fecundity in
female S. ratti was limited by availability of sperm, using
two mating regimes. Firstly, 10 virgin free-living male and
female S. ratti were allowed to mate overnight on plates and
the males were removed the next day; secondly, 20 virgin
free-living female S. ratti and 10 virgin male S. ratti were
allowed to mate overnight and the old males removed and
10 new ones added daily. Non-parametric Wilcoxon
analyses (using c2 test) were used to compare life-time
fecundity in the two treatments and life-time fecundity
across temperature. The effect of age on daily fecundity was
tested using linear regression analysis.
2.5. Pharyngeal pumping rate
This was measured during aging in free-living
S. ratti females as for C. elegans (Kenyon et al., 1993;
Gems et al., 1998). A single pump was scored as a complete
backward movement of the terminal bulb of the pharynx,
viewed at 40! magnification, using a Zeiss Stemi 2000-C
stereomicroscope (Keane and Avery, 2003). Free-living
S. ratti females were maintained under optimised culture
conditions (25 8C) and each day over a 4-day-period, 20
randomly selected nematodes were placed on separate
plates, allowed to acclimatise for one minute and then for
each pumping worm, the number of pumps in two, one
minute intervals was counted. From this, the average
pumping rate and the percentage of animals pumping each
day was calculated. If no pumping was seen in either one
minute scoring interval, the worm was scored as nonpumping. The effect of age on the mean pharyngeal
pumping rate and on the percentage of animals pumping
was tested using linear regression analysis.
2.6. Movement
L4 S ratti females were placed on each of eight plates
and individual L4 S. ratti females were placed on 40
plates and maintained under optimised culture conditions
(25 8C). The movement of grouped and individual freeliving females was observed daily and classified as for
C. elegans (Herndon et al., 2002). Class A worms move
voluntarily and leave sinusoidal tracks, class B worms
only move when prodded and leave non-sinusoidal tracks
and class C worms do not move forward or backward
even when prodded. Non-parametric Wilcoxon analyses
(using c2 test) were used to compare percentages of class
A, B and C of grouped worms on each day, to test
whether there was an age-related decline in movement of
S. ratti. The analysis of individual worms tested whether
there was variation in the timing of any age-related
decline in movement.
2.7. Autofluorescence
Daily microfluorometric measurements of free-living
S. ratti females aged 1–6 days (19 8C) were taken using
a Leica DMRXA2 microscope using either a narrowband
DAPI filter set (excitation: 340–380 nm), or a GFP filter
set (excitation: 450–490 nm). In aqueous C. elegans
extracts there is a peak of fluorescence with an excitatory
wavelength range 330–350 nm, and an emission peak at
430–460 nm (32). This blue fluorescence, which increases
in intensity with age, is visualised using the DAPI filter
set. The GFP filter was employed since use of a similar
filter set revealed age increases in autofluorescence in
C. elegans (Garigan et al., 2002). Images at 100!
magnification were captured using a Hamamatsu OrcaER
digital camera, and pixel density of selected regions of
images of fluorescent nematodes measured using the
Openlab 3.1.4 (Improvision, www.improvision.com)
image analysis software. Specifically, for each nematode,
M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
1271
the most anterior region of the gut in which wellfocused, punctate autofluorescent granules were visible
was selected and images captured with the same
exposure, which avoid pixel saturation. Sometimes
distension of the anterior intestine led to diffuse
fluorescence, in which case a more posterior region of
intestine was selected. In some 6-day-old animals in
which the intestine was not clearly distinct, these were
excluded from study. For both DAPI and GFP filters,
autofluorescence was measured in R16 animals apart
from day six, when 12 animals were used. The effect of
age on fluorescence was tested using linear regression
analysis.
2.8. Microscopic examination of aging nematodes
Appearance of major anatomical features was compared
in one and 4-day-old S. ratti females using Nomarski
(differential interference contrast) microscopy, with magnification up to 1000!, using standard C. elegans methods
(Schnabel, 1999).
3. Results
3.1. Optimisation of culture conditions for S. ratti survival
In contrast to C. elegans, it is not possible to prepare
cultures of S. ratti that are monoxenic (i.e. with E. coli
alone) since larvae are isolated directly from rodent faeces.
To attempt to minimise microbial contamination from the
latter (which might otherwise reduce survival), S. ratti was
cultured on agar plates with streptomycin-resistant E. coli
OP50 (E. coli SR) and a range of concentrations of
streptomycin (50, 200, 400, 700 or 1000 mg/ml). Under
these conditions, visible contamination by other microbes
was reduced but not prevented. Maximum mean life-span
was 4.2G0.2 days at 400 mg/ml streptomycin which was
significantly greater than on E. coli OP50 without antibiotic
(mean lifespan: 3.7G0.2 days; c2Z11.4, PZ0.0007;
Fig. 2A).
In C. elegans, prevention of bacterial proliferation leads
to extended lifespan, showing that lifespan is shortened by
the E. coli food source (Garigan et al., 2002). We explored
whether this might be the case in S. ratti. However, mean
lifespan of virgin females under optimised conditions
(400 mg/ml streptomycin, E. coli SR) was greater (4.1G
0.2 days) than on non-resistant E. coli to which had been
added 200 mg/ml streptomycin (2.4G0.1 days; c2Z50.7,
P!0.0001), 20 mg/ml kanamycin (2.9G0.2 days; c2Z24.6,
P!0.0001) or 12.5 mg/ml tetracyclin (3.2G0.1 days; c2Z
17.6, P!0.0001).
To explore whether S. ratti survives better if cultured
on bacteria found in their natural environment, it was
cultured on either of two bacterial isolates from rat faeces,
identified as Bacillus spp. and S. faecalis. Mean lifespan
Fig. 2. Factors affecting S. ratti lifespan. (A) Survivorship of virgin freeliving females under optimised culture conditions (400 mg/ml streptomycin
and E. coli SR) (x, —) and on E. coli OP50 (o, - - -). (B) Effects of
temperature on mean (o, - - -) and maximum (x, —) lifespan of virgin freeliving females. S. ratti adults die within 24 h at 37 8C. (C) Effect of
temperature on lifetime fecundity per animal of S. ratti females, mating on
day one only (o, - - -) and mating throughout life (x, —).
proved to be longer on E. coli SR with 400 mg/ml
streptomycin (4.2G0.2 days) than on either Bacillus
spp. (1.7G0.1 days; c2Z89.1, P!0.0001) or S. faecalis
(1.5G0.1 days; c2Z96.0, P!0.0001).
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M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
Table 1
The effect of sex-ratio on mean lifespan of female and male S. ratti (19 8C)
Animals per plate
Males
Females
0
20
40
60
80
100
80
60
40
20
Male/
female ratio
0
0.25
0.67
1.5
4.0
Mean lifespan (daysGSE)
Females
Males
2.83G0.19
2.13G0.22
2.52G0.28
3.13G0.39
3.18G0.40
–
2.89G0.42
2.33G0.21
1.79G0.17
1.69G0.13
3.2. Environmental and behavioural determinants
of fecundity and lifespan
To test the effect of sex-ratio on free-living male and
female S. ratti lifespan, L4 worms of each sex were
maintained on plates (100 animals/treatment, 25 animals/plate) at different male: female ratios. As the ratio of males
to females increased, there was no affect on female lifespan
(Spearman’s rank correlation, rZ0.7, PZ0.19) but male
lifespan decreased (Spearman’s rank correlation, rZK1.0,
P!0.0001) (Table 1), suggesting that mating aids survival
in S. ratti males.
To test the effect of population density on lifespan, virgin
free-living male S. ratti were maintained at densities of 1,
25, 50 and 100 animals/plate and virgin free-living females
at densities of 1, 20, 50 and 100 animals/plate. For
C. elegans, lifespan is reduced by increased population
density in males but not hermaphrodites (Gems and Riddle,
2000). In S. ratti, female lifespan did not differ between
female densities of 1 and 50 animals per plate (c2Z0.57,
PZ0.43), but it did between densities of 1 and 100 animals
per plate (1 vs. 100: (c2Z45.2, P!0.0001; Table 2).
Likewise, male lifespan did not differ between densities of 1
and 50 animals per plate (c2Z3.6, PZ0.06), but it did
between densities of 1 and 100 animals per plate (1 vs. 100:
c2Z9.6, PZ0.002; Table 2).
To test the effect of temperature on lifespan of virgin
free-living S. ratti females, lifespan was measured at 4, 10,
15, 19, 25, 30 and 37 8C, with 20 worms per plate and 100
worms per treatment. There was a significant reduction in
both mean and maximum lifespan with increasing temperature over the range 10–30 8C (mean: F1,3Z78.1, PZ0.003;
maximum: F1,3Z196.7, PZ0.0008; Fig. 2B).
Lifetime fecundity was higher when the females
were supplied with fresh, virgin males each day at 15, 19
Table 2
Effect of density on mean lifespan of virgin S. ratti (19 8C)
Population density
(animals/plate)
Females
Males
1
20, 25a
50
100
3.09G0.21
3.11G0.17
3.14G0.19
1.67G0.09
2.07G0.15
1.91G0.09
2.02G0.09
1.62G0.08
a
Mean lifespan (daysGSE)
Density of 20 animals/plate for females, 25 animals/plate for males.
and 25 8C (c2Z4.9, PZ0.028; c2Z6.9, PZ0.009; c2Z
4.8, PZ0.028, respectively) (Fig. 2C). Daily fecundity
declines with age at both 19 and 25 8C (F1,4Z15.5, PZ
0.017; F1,2Z35.0, PZ0.027, respectively) and lifetime
fecundity varies significantly with temperature (c2Z18.9,
P!0.0001; Fig. 2C) and at 25 8C was significantly greater
than at any other temperature (data not shown). Therefore,
by this criterion, 25 8C is closest to the optimal temperature
for fitness. However, under these conditions, the maximum
lifetime fecundity was less than 40 progeny per female,
compared with over 1400 in C. elegans (Hodgkin and
Barnes, 1991).
Interestingly, there was a decrease in post-reproductive
lifespan with increasing temperature. As a percentage of
maximum lifespan, this was 50% at 10 8C, 56% at 15 8C,
25% at 19 8C, 0% at 25 8C and 30 8C. This contrasts with
C. elegans, where there is a large (e.g. 60% at 20 8C) postreproductive lifespan that is not affected by temperature
(Klass, 1977; Gems, 2002).
3.3. Analysis of age-specific mortality
Mean and maximum lifespans for S. ratti and for
C. elegans hermaphrodites maintained under the same
conditions and C. elegans hermaphrodites maintained in
monoxenic conditions are given in Table 3. The mean
lifespan of C. elegans in either culture condition was
significantly longer than that of S. ratti (same conditions:
c2Z1074.0, P!0.0001; monoxenic: c2Z1201.0, P!
0.0001) (Fig. 3A). C. elegans mean lifespan was greater
in monoxenic culture conditions than in S. ratti optimised
culture conditions (c2Z529.0, P!0.0001), presumably due
to the presence of contaminating microbes in the S. ratti
culture conditions (Fig. 3A).
Limitation of lifespan by aging, rather than some other
cause of mortality, may be established by testing for the
occurrence of an exponential increase in mortality rate with
increasing age (Wachter and Finch, 1997). In all these three
experiments an age-specific mortality rate acceleration was
observed. There was a significant positive linear relationship between ln (m(t)) and age for S. ratti and C. elegans
under optimised culture conditions (S. ratti: F1,14Z86.3,
P!0.0001; C. elegans: F1,16Z45.7, P!0.0001) and for
C. elegans cultured monoxenically (F1,23Z88.8, P!0.0001
(Fig. 3B). The MRDT is significantly lower in S. ratti
females than in C. elegans hermaphrodites, whether culture
conditions in the latter were the same, or different to S. ratti
(tZ3.3, P!0.01; tZ3.8, P!0.001, respectively) (Table 3).
There was no difference in the MRDT for C. elegans under
the two culture conditions (tZ2.0, PO0.05). The IMR is
greater in S. ratti females than in C. elegans hermaphrodites
under either the same or different culture conditions (tZ2.0,
P!0.001; tZ13.8, P!0.001, respectively) (Table 3). The
IMR for C. elegans kept under S. ratti culture conditions is
almost twice that of the monoxenically cultured populations
(tZ5.0, P!0.001) (Table 3). Overall, these results imply
M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
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Table 3
Mean and maximum lifespan, initial mortality rate (IMR) and mortality rate doubling time (MRDT) at 25 8C for virgin free-living female S. ratti and
hermaphrodite C. elegans at optimum S. ratti conditions (1) and under monoxenic conditions (2)
Species
S. ratti
C. elegans
C. elegans
Condition
1
1
2
LifespanGSE (days)
Mean
Maximum
3.0G0.1
7.7G0.1
11.2G0.1
4.5G0.8
11.5G2.1
18.3G4.3
that S. ratti is shorter lived than C. elegans because (a) it is
more frail (as reflected by higher IMR), and (b) it ages more
quickly (as reflected by a lower MRDT).
Using the mortality parameters in Table 3, it is possible
to estimate what S. ratti lifespan might be, if it were possible
to culture it monoxenically. For C. elegans, only the IMR
differed significantly between the two culture conditions
and was lower in monoxenic culture conditions. On
monoxenic culture, C. elegans IMR is reduced by 50%
relative to optimal S. ratti culture conditions. Substituting
an S. ratti IMR reduced by a similar proportion into the
equation ln (m(t)) against age, gives estimated median and
maximum lifespans of monoxenically cultured S. ratti of
4.6G0.8 and 5.3G1.0 days, respectively. Alternatively,
substituting the lower C. elegans IMR value into the
equation ln (m(t)) against age, gives an upper estimate to
median and maximum lifespans of monoxenically cultured
S. ratti of 6.8G1.5 and 7.7G1.7 days, respectively. These
contrast with observed median and maximum lifespans of
3.6G0.6 and 4.5G0.8 days, respectively.
IMRGSE (10K3 daysK1)
MRDTGSE (days)
25G2
3.6G0.3
1.8G0.2
0.84G0.09
1.42G0.15
2.01G0.30
there were only class C worms remaining. Analysis of agerelated changes in movement for 40 individual females
found that of 32 class A animals, 26 proceeded through
stages B and C before death, four became class C animals
directly from class A and two died after becoming class B
animals. Likewise, of 36 class B animals, 33 proceeded
through stage C before dying.
We examined autofluorescence levels, which in
C. elegans increase with age (Davis et al., 1982; Garigan
et al., 2002). Microfluorometric measurements on one to
3.4. Changes accompanying aging
First we examined age-related changes in feeding
behaviour. In C. elegans, the rate of pharyngeal pumping
declines gradually with age (Kenyon et al., 1993). In S. ratti,
there was a reduction in both the rate of pharyngeal pumping
(F1,48Z130.0, P!0.0001) and in the percentage of live
animals pumping (F1,2Z62.3, PZ0.016) (Fig. 4A) with age,
consistent with typical nematode aging (Gems, 2002).
Second we examined age-related changes in movement.
As with C. elegans (Herndon et al., 2002), S. ratti could be
classified as class A (moving voluntarily), class B (only
moving when prodded) or class C (not moving away but
alive) behavioural phenotypes. There was an age-associated
decline in movement (Fig. 4B). Specifically, on days 1 and 2
there were greater percentages of class A than class B
worms (day 1: c2Z11.4, P!0.001; day 2: c2Z6.0, PZ
0.014) and greater percentages of class B than class C
worms (day 1: c2Z4.7, PZ0.03; day 2: c2Z5.3, PZ
0.021). On day 3 there were no differences in the
percentages of the three behavioural classes (A vs. B:
c2Z2.6, PZ0.110; B vs. C: c2Z0.05, PZ0.827; A vs. C:
c2Z3.0, PZ0.083), but by day 4 classes B and C were
predominating (A vs. B: c2Z7.6, PZ0.006; B vs. C: c2Z
3.3, PZ0.068; A vs. C: c2Z9.6, PZ0.002) and by day 5
Fig. 3. (A) Survival and (B) plots of ln (m(t)) against age for S. ratti (x, —)
and C. elegans (o, —) under optimised culture conditions and C. elegans
(C, —) in monoxenic cultures.
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M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
six day old animals revealed significant increases in
autofluorescence viewed using both DAPI (F1,105Z18.2,
P!0.0001) (Fig. 4C–E) and GFP filters (F1,109Z11.8,
P!0.001) (Fig. 4C).
Next we examined age changes in the appearance of the
major nematode organs, comparing 1-day-old and 4-dayold adults observed with Nomarski microscopy (4-day-old
adults, NZ8). Overall, the appearance of aging S. ratti
adults appeared similar to that of aging C. elegans, as
previously described (Garigan et al., 2002; Herndon et al.,
2002). In the main, the gross structural integrity of the major
organs (e.g. the cuticle and reproductive system) was well
preserved in older animals. However, in the pharynx, the
majority of four day old animals showed swelling of the
proporcus and metacorpus, which was often accompanied
by the presence of large quantities of bacteria (Fig. 4G).
Microbial blockage of the buccal cavity and pharynx has
also been observed in aging C. elegans (Garigan et al.,
2002). The most striking morphological changes occurred in
the intestine. Whilst in 1-day-old animals there were large,
healthy looking refractile intestinal cells (Fig. 4H), in the
majority of four day old animals these cells were markedly
atrophied and had a ragged appearance (Fig. 4I).
4. Discussion
In this study we document the phenomenology of aging
in the very short-lived, free-living adults of the parasitic
nematode S. ratti, comparing it with the longer-lived, wellcharacterised free-living nematode species C. elegans. One
question that we wished to address was: is the short lifespan
of S. ratti adults due to very rapid aging? Our results show
clearly that it is.
This conclusion is supported by a number of observations. Most significantly, S. ratti exhibits an acceleration
in age-specific mortality rate, one which is more rapid than
that seen in C. elegans (Fig. 3B, Table 3). Several
characteristics of organismal aging previously documented
in C. elegans also occur in S. ratti, but at a faster rate. For
example, with increasing age, levels of autofluorescence
(lipofuscin) increase (Fig. 4C–E) and the rate of feeding
3
Fig. 4. (A) Mean pharyngeal pumping rate (pumps per second) (o, - - -) and
percentage of live animals pumping (x, —) with age for female S. ratti at
25 8C. (B) Percentage of grouped worms of class A (dark grey), class B
(light grey), class C (white) and dead worms (black) with age for female
S. ratti at 25 8C. (C) Changes with age in autofluorescence viewed using
DAPI (x, —) and GFP (o, - - -) filters in female S. ratti at 19 8C. (D–E)
Increased autofluorescence during aging in S. ratti free-living females
viewed with a DAPI filter; (D) one day old adult; (E) four day old adult.
BarsZ200 mM. (F–I) S. ratti free-living adults viewed using differential
interference contrast (Nomarski) microscopy. (F) and (H) one day old
adults; (G) and (I), four-day-old adults. (F) and (G) Pharyngeal region. In
(F), the entire pharynx is visible, in (G), the procorpus (P) and metacorpus
(MC) of the pharynx are distended and packed with bacteria (B). (H) and (I):
posterior intestine. In (H), note the large, healthy refractile intestinal cells
(I). In (I), the intestine is strikingly atrophied. Anus (AN). BarsZ20 mM.
M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
(Fig. 4A) and movement (Fig. 4B) decline. We observed an
age-related decline in movement of S. ratti. In C. elegans a
wide variation in the time of onset of reduction in movement
was observed that supported the existence of substantial
stochasticity in this trait. In S. ratti there was a less marked
variation in time of onset of movement reduction, though
about 20% of class A animals did die directly, consistent
with some stochasticity. S. ratti lifespan and lifetime
fertility are also affected by temperature (Fig. 2B and C)
in a manner similar to that seen in C. elegans (Klass, 1977).
Hence, the extreme brevity of life in free-living S. ratti
adults, the shortest lived nematode thus far described, is the
consequence of rapid aging, rather than some other, more
swift and catastrophic life-shortening pathology.
Some aspects of aging in S. ratti differed from those in
C. elegans. For example, in S. ratti, an increase in the ratio
of males to females did not decrease female lifespan,
whereas it does in C. elegans (Gems and Riddle, 1996), and
in some other nematode species, e.g. Ditylenchus triformis
(reviewed in Gems, 2001). In C. elegans this appears to be
due to the effects of copulation rather than increased egg
production (Gems and Riddle, 1996). In S. ratti, male
lifespan was actually increased by exposure to females,
whereas in C. elegans, mating greatly reduces male lifespan
(Gems and Riddle, 2000). In the terrestrial nematode
Panagrellus redivivus, mating shortens lifespan in females
(as in C. elegans) but increased it in males (as in S. ratti). In
C. elegans, grouping of males results in the formation of
homosexual mating clumps, which approximately halves
male lifespan compared with solitary males (Gems and
Riddle, 2000). However, no such deleterious effects of
male-male interactions were seen in S. ratti males, perhaps
consistent with the absence of any observed clumping
behaviour.
Why is the lifespan of this organism so short? In general,
terrestrial nematodes such as C. elegans are very short lived,
with maximum lifespans of the order of weeks to months.
Among the longest lived is Rhabditis tokai, with a
maximum lifespan of 145 days (Suzuki et al., 1978). By
contrast, parasitic nematodes evolve much longer lifespans,
with some hookworm and filarial nematode species
attaining ages of more than a decade. Free-living S. ratti
adult females are genetically identical to the adult parasitic
females, yet the maximum lifespan of the latter is at least 11
months (Gemmill et al., 1997; Gardner, Gems and Viney,
unpublished). Potentially, these evolved differences may
reflect the differences in extrinsic mortality experienced by
exposed free-living versus host-protected parasitic species.
Yet it remains unclear why the lifespan of free-living S. ratti
females is so much shorter that those of other terrestrial
nematodes without parasitic morphs.
In conclusion, here we have documented age-changes in
the free-living S. ratti morph; the shortest-lived nematode
thus far described and one of the shortest-lived animal
species, whose brevity of life appears to be the consequence
of extremely rapid aging.
1275
Acknowledgements
We thank Louise Phelan, Sadie Iles-Ryan and Clare
Wilkes for excellent technical support, Geoff Werrett for
microbiology work and Will Mair for reading the manuscript. MPG is supported by a grant to MEV and DG from
the Experimental Research on Ageing Programme of the
BBSRC and we thank them for their support. MEV is also
supported by the MRC, NERC and the Wellcome Trust. DG
is also supported by funds from the EU (Framework V), the
Wellcome Trust, and the Royal Society. C. elegans and
E. coli OP50 strains were supplied by the Caenorhabditis
Genetics Center which is supported by the National
Institutes of Health, National Center for Research
Resources, USA.
References
Austad, S.N., 1993. Retarded senescence in an insular population of
Virginia opossum (Didelphis virginiana). J. Zool. 229, 695–708.
Austad, S.N., Fischer, K.E., 1991. Mammalian aging, metabolism, and
ecology: evidence from the bats and marsupials. J. Gerontol. A Biol.
Sci. Med. Sci. 46, B47–B53.
Chapuisat, M., Keller, L., 2002. Division of labour influences the rate of
ageing in weaver ant workers. Proc. R. Soc. Lond. Ser. B 269, 909–913.
Comfort, A., 1979. The Biology of Senescence, 3rd ed Elsevier, New York.
Cowan, S.T., Barrow, G.I., Feltham, R.K.A., Steel, K.J., 1993. Cowan and
Steel’s manual for the identification of medical bacteria. Cambridge
University Press, Cambridge.
Davis, B.O., Anderson, G.L., Dusenbery, D.B., 1982. Total luminescence
spectroscopy of fluorescence changes during aging in Caenorhabditis
elegans. Biochemistry 21, 4089–4095.
Edney, E.B., Gill, R.W., 1968. Evolution of senescence and specific
longevity. Nature 220, 281–282.
Finch, C.E., 1990. Longevity, Senescence and the Genome. University of
Chicago Press, Chicago.
Garigan, D., Hsu, A.-L., Fraser, A.G., Kamath, R.S., Ahringer, J.,
Kenyon, C., 2002. Genetic analysis of tissue aging in Caenorhabditis
elegans: a role for heat-shock factor and bacterial proliferation.
Genetics 161, 1101–1112.
Garsin, D., Villanueva, J., Begun, J., Kim, D., Sifri, C., Calderwood, S.B.,
Ruvkun, G., Ausubel, F.M., 2003. Long-lived C. elegans daf-2 mutants
are resistant to bacterial pathogens. Science 300, 1921.
Gemmill, A.W., Viney, M.E., Read, A.F., 1997. Host immune status
determines sexuality in a parasitic nematode. Evolution 51, 393–401.
Gems, D., 2001. Longevity and ageing in parasitic and free-living
nematodes. Biogerontology 1, 289–307.
Gems, D., 2002. Ageing, in: Lee, L.D. (Ed.), The Biology of Nematodes.
Taylor and Francis, London, pp. 413–455.
Gems, D., Riddle, D.L., 1996. Longevity in Caenorhabditis elegans
reduced by mating but not gamete production. Nature 379, 723–725.
Gems, D., Riddle, D.L., 2000. Genetic, behavioural and environmental
determinants of male longevity in Caenorhabditis elegans. Genetics
154, 1597–1610.
Gems, D., Sutton, A.J., Sundermeyer, M.L., Albert, P.S., King, K.V.,
Edgley, M.L., Larsen, P.L., Riddle, D.L., 1998. Two pleiotropic classes
of daf-2 mutation affect larval arrest, adult behavior, reproduction and
longevity in Caenorhabditis elegans. Genetics 150, 129–155.
George, J., Bada, J., Zeh, J., Scott, L., Brown, S., O’Hara, T., Suydam, R.,
1999. Age and growth estimates of bowhead whales (Balaena
mysticetus) via aspartic racemization. Can. J. Zool. 77, 571–580.
1276
M.P. Gardner et al. / Experimental Gerontology 39 (2004) 1267–1276
Harvey, S.C., Gemill, A.W., Read, A.F., Viney, M.E., 2000. The control of
morph development in the parasitic nematode Strongyloides ratti. Proc.
R. Soc. Lond. Ser. B 267, 2057–2063.
Herndon, L.A., Schmeissner, P.J., Dudaronek, J.M., Brown, P.A.,
Listner, K.M., Sakano, Y., Paupard, M.C., Hall, D.H., Driscoll, M.,
2002. Stochastic and genetic factors influence tissue-specific decline in
ageing C. elegans. Nature 419, 808–814.
Hodgkin, J., Barnes, T.M., 1991. More is not better: brood size and
population growth in a self-fertilizing nematode. Proc. R. Soc. Lond.
Ser. B 246, 19–24.
Keane, J., Avery, L., 2003. Mechanosensory inputs influence Caenorhabditis elegans pharyngeal activity via ivermectin sensitivity genes.
Genetics 164, 153–162.
Kenyon, C., Chang, J., Gensch, E., Rudener, A., Tabtiang, R.A., 1993. A C.
elegans mutant that lives twice as long as wild type. Nature 366,
461–464.
Klass, M.R., 1977. Aging in the nematode Caenorhabditis elegans: major
biological and environmental factors influencing life span. Mech.
Ageing Dev. 6, 413–429.
Klass, M.R., Hirsh, D.I., 1976. Non-ageing development variant of
Caenorhabditis elegans. Nature 260, 523–525.
Lakowski, B., Hekimi, S., 1998. The genetics of caloric restriction
in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 95, 13091–13096.
Lyman, C.P., O’Brien, R.C., Greene, G.C., Papafrangos, E.D., 1981.
Hibernational longevity in the Turkish hamster Mesocricetus brandti.
Science 212, 668–670.
Martı́nez, D.E., 1998. Mortality patterns suggest lack of senescence in
hydra. Exp. Gerontol. 33, 217–225.
McCay, C., Crowell, M., Maynard, L., 1935. The effect of retarded growth
upon the length of the life span and upon the ultimate body size. J. Nutr.
10, 63–79.
Nusbaum, T., Rose, M., 1999. The effects of nutritional manipulation and
laboratory selection on lifespan in Drosophila melanogaster.
J. Gerontol. A Biol. Sci. Med. Sci. 54, B192–B198.
Page, R.E., Peng, C.Y.-S., 2001. Aging and development in social insects
with emphasis on the honey bee, Apis mellifera L. Exp. Gerontol. 36,
695–711.
Roth, G.S., Lane, M.A., Ingram, D.K., Mattison, J.A., Elahi, D.,
Tobin, J.D., Muller, D., Metter, E.J., 2002. Biomarkers of caloric
restriction may predict longevity in humans. Science 297, 811.
Schnabel, R., 1999. Microscopy, in: Hope, I.A. (Ed.), C. elegans, A
Practical Aproach. Oxford University Press, Oxford, pp. 119–141.
Sherman, P., Jarvis, J., 2002. Extraordinary life spans of naked mole-rats
(Heterocephalus glaber). J. Zool. 258, 307–311.
Sohlenius, B., 1969. Studies on the population development of Mesodiplogaster biformis (Nematoda, Rhabditida) in agar culture. Pedobiologia 9, 243–253.
Spieler, M., Schierenberg, E., 1995. On the development of the alternating
free-living and parasitic generations of the nematode Rhabdias bufonis.
Invertebr. Reprod. Dev. 28, 193–203.
Sulston, J., Hodgkin, J., 1988. Methods, in: Wood, A.B. (Ed.), The
Nematode Caenorhabditis elegans. Cold Spring Harbor Press, New
York, pp. 587–606.
Suzuki, K., Hyodo, M., Ishii, N., Moriya, Y., 1978. Properties of a strain of
free-living nematode, Rhabditidae sp.: life cycle and age-related
mortality. Exp. Gerontol. 13, 323–333.
Viney, M.E., 1994. A genetic analysis of reproduction in Strongyloides
ratti. Parasitology 109, 511–515.
Viney, M.E., 1996. Developmental switching in the parasitic nematode
Strongyloides ratti. Proc. R. Soc. Lond. Ser. B 263, 201–208.
Viney, M.E., Matthews, B.E., Walliker, D., 1993. Mating in the nematode
parasite Strongyloides ratti: proof of genetic exchange. Proc. R. Soc.
Lond. Ser. B 254, 213–219.
Wachter, K.W., Finch, C.E., 1997. Between Zeus and the Salmon. National
Academy Press, Washington DC.
Wilkinson, G., South, J., 2002. Life history, ecology and longevity in bats.
Aging Cell 1, 124–131.
Williams, G.C., 1957. Pleiotropy, natural selection and the evolution of
senescence. Evolution 11, 398–411.
Yamada, M., Matsuda, S., Nakazawa, M., Arizona, N., 1991. Speciesspecific differences in heterogonic development of serially transferred
free-living generations of Strongyloides planiceps and Strongyloides
stercoralis. J. Parasitol. 77, 592–594.